Method and device employing covalently immobilized colored dyes

Abstract

A test method and device employ a matrix to which is covalently bonded a second component of a two component dye system. Color is formed when the first component, which may be the analyte or another dye component, covalently couples to the second component. The color formed is covalently immobilized to the matrix. A preferred second component, 5-amino-2-naphthalene sulfonic acid, is covalently bonded via the amino group and forms a colored covalent adduct with oxidized 4-aminoantipyrine. Another preferred second component is diazotized para-aminoaniline, which is covalently bonded via the amino group and forms a colored covalent adduct directly with bilirubin.

What is claimed is: 
     
       1. A method of qualitatively or quantitatively determining the presence or amount of an analyte in a test sample, comprising the steps of: a) contacting a solid phase matrix to a liquid phase, said solid phase matrix having covalently bound thereto a second component of a two component dye system, and   said liquid phase containing a first component of said dye system, the amount of said first component being equal to or proportional to the amount of analyte present in the test sample,     whereby said second component further covalently binds said first component thereby forming a permanently colored covalent adduct that ramains covalently bound to said solid phase matrix; and   b) determining the presence or amount of said colored covalent adduct on said solid phase matrix as a measure of the presence or amount of the analyte in the test sample.   
     
     
       2. The method according to claim 1 wherein the analyte is a peroxide. 
     
     
       3. The method according to claim 1 wherein the first dye component is the analyte. 
     
     
       4. The method according to claim 1 wherein said first and second components are initially colored and covalently bind to form a distinguishable colored compound. 
     
     
       5. The method according to claim 1 wherein said first and second components are initially colorless and covalently bind to form a colored compound. 
     
     
       6. The method according to claim 1 wherein said first component is generated by a reagent means in an amount proportional to the amount of analyte in the test sample. 
     
     
       7. The method according to claim 1 wherein a reagent means renders said first component covalently coupleable to said second component in an amount proportional to the amount of analyte in the test sample. 
     
     
       8. The method according to claim 7 wherein said reagent means is an oxidase-peroxidase enzyme system. 
     
     
       9. The method according to claim 1 wherein said solid phase matrix is a chromatographic strip through which said liquid phase advances from an origin to a terminus, and wherein said colored dye compound is produced on said strip in the form of a color bar, the length of said color bar being proportional to the amount of analyte present in said test sample. 
     
     
       10. The method according to claim 1 wherein said second component is covalently bound to said solid phase matrix by activating reactive groups on at least one of said solid phase matrix and said second component and covalently binding said second component to said solid phase matrix via said activated reactive groups. 
     
     
       11. The method according to claim 1, wherein said matrix is a chromatographic strip and the presence or amount of the analyte in the test sample is proportional to the length of said colored covalent adduct immobilized along the length of said strip. 
     
     
       12. The method according to claim 1, wherein the amount of the analyte in the test sample is proportional to the intensity of said colored covalent adduct immobilized on said matrix. 
     
     
       13. A method of qualitatively or quantitatively determining the presence or amount of an analyte in a test sample, comprising the steps of: a) contacting a solid phase matrix to a liquid phase, said solid phase matrix having covalently bound thereto a second component of a two component dye system, said second component selected from the group consisting of para-unsubstituted phenolic components, para-unsubstituted aromatic amine compounds and para-halogenated phenolic or aromatic amine compounds, and   said liquid phase containing a first component of said dye system, the amount of said first component being equal to or proportional to the amount of analyte present in the test sample,   whereby said second component further covalently binds said first component thereby forming a permanently colored covalent adduct that remains covalently bound to said solid phase matrix; and     b) determining the presence or amount of said colored covalent adduct on said solid phase matrix as a measure of the presence or amount of the analyte in the test sample.   
     
     
       14. The method according to claim 13, wherein said first component is 4-aminoantipyrine. 
     
     
       15. The method according to claim 13, wherein said second component is 5-amino-2-naphthalene sulfonic acid. 
     
     
       16. The method according to claim 13, wherein said matrix is a chromatographic strip and the presence or amount of the analyte in the test sample is proportional to the length of said colored covalent adduct immobilized along the length of said strip. 
     
     
       17. The method according to claim 13, wherein the amount of the analyte in the test sample is proportional to the intensity of said colored covalent adduct immobilized on said matrix. 
     
     
       18. A method of qualitatively or quantitatively determining the presence or amount of an analyte in a test sample, comprising the steps of: a) contacting a chromatographic strip to a liquid phase, said strip having covalently bound thereto a second component of a two component dye system, and   said liquid phase containing the analyte which is a first component of said dye system, and a reagent means which renders the analyte reactive with said second component,   whereby said second component covalently binds said reactive analyte thereby forming a permanently colored covalent adduct that remains covalently bound to said strip; and     b) determining the presence or amount of said colored covalent adduct on said strip as a measure of the presence or amount of the analyte in the test sample.   
     
     
       19. The method according to claim 18, wherein the presence or amount of the analyte in the test sample is proportional to the length of said colored covalent adduct immobilized along the length of said strip. 
     
     
       20. The method according to claim 18, wherein the amount of the analyte in the test sample is proportional to the intensity of said colored covalent adduct immobilized on said matrix.